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Affymetrix microarray

What is Affymetrix microarray?

https://www.cgs.path.cam.ac.uk/arrays/gene-expression/Affymetrix
https://www.thermofisher.com/de/de/home/life-science/microarray-analysis/affymetrix.html
The purpose of Affymetrix microarray?
Purpose of the method and what
does it entail?

● DNA microarrays have paved way for scientists in


identification and mapping of diseases
● Large-scale experiments, which allows for scientists to predict
disease progression and assign function to previously
unannotated genes, can be conducted using microarrays
● Researchers may use microarrays to compare gene
expression in two different cell types or tissue samples, e.g.
healthy and diseased tissue
https://es.wikipedia.org/wiki/Archivo:Microarray-schema.gif
Key Applications
● Human genotyping
○ Has enabled identification of thousands of associations between genetic
variants and diseases or traits, an created maps of unique variation within
populations.
● Transcriptome analysis
○ Attempts to catalogue and quantify the RNA content of a cell, tissue or
organism. In some cases, the goal is to target all transcripts, regardless of
their structure or function.
● Cytogenetics analysis
○ Deals with the study of the complete human genome arranged in the form of
46 chromosomes. DNA microarray has the capability to study almost the entire
human genome on a single chip.
● miRNA profiling
○ Measuring the changes in the miRNA expression profile is extremely important
for deciphering the biological context of differentially expressed genes.
https://www.frontiersin.org/articles/10.3389/fevo.2014.00031/full
How does it work?
The microarray

● A collection of DNA probes attached to a solid support (cartridge or a plate).


● The probes and their positions are known.
● Many different arrays for humans, mice, cancer panels, etc are on the market
● The probes can be custom-made and prepared in two ways:
○ Prepared beforehand and attached to the surface.
○ Chemically synthesised directly on it.
Photolithography - a series of masks and mirrors de-protect certain
nascent oligonucleotides using light.
○ Ink-jet depositions - print one of the four DNA bases where you need it to
be.
The sample
● Comparative method - two samples are needed (example: healthy vs cancerous cells).
Final product
● Hybridization - formation of double-stranded nucleic
acids which can reassemble with perfect fidelity.
● Use lasers to analyse/scan the array.
● Different sample, different labels, different colour
signal.
● If signal present, gene was expressed in the sample.
● Microarray analysis isn’t very quantitative. Highest and
lowest intensities exist, but it’s a relative method, only
a ratio is known.
Advantages
● relative array: even small sample sizes can be detected
● statistical rigour: photochemical synthesis allows inclusion of multiple probes
● highly standardised
● variety of different arrays available; customisation possible
● diverse range of applications
● high correlation between microarrays and sequencing
● fast, cost-effective
Disadvantages
● Cross-hybridisation can interfere with results
● Relative method, so measured intensities have to be compared
● Hard to detect alternative splicing
● Few producers of necessary materials
● Microarrays are becoming less popular therefore technical support might dwindle
● Low abundance transcripts are difficult to detect
● Comparing expression levels across different experiments can require complicated
normalisation methods
● Can be expensive
The use of Affymetrix in research
Increase expression of CD177 in Kawasaki disease

The article:

- Taiwanese study
- Published in April 2019 in the Pediatric
Rheumatology online journal
- Authors: Ying-Hsien Huang; Mao-Hung
Lo; Xin-Yuan Cai; Shih-Feng Liu; Ho-
Chang Kuo

→ How genes may contribute to the


pathogenesis of Kawasaki disease.

Huang Y-H, Lo M-H, Cai X-Y, Liu S-F, Kuo H-C.


Increase expression of CD177 in Kawasaki
disease. Pediatric Rheumatology. 2019;17(1).
Kawasaki disease
Kawasaki disease (KD) is a disease in which blood vessels throughout the body
become inflamed that occurs predominantly in infants and young children.

Kawasaki disease is rare. It affects between 8 and 67 per 100,000. It is much less
common after the age of five. The disorder was first described in 1967 by
Tomisaku Kawasaki in Japan.

Its cause is unknown. An important genetic contribution to disease susceptibility


is suggested by the higher incidence among U.S. children of Asian/Pacific Islander
descent in Hawaii and California (210 and 50.4 per 100,000, respectively).
How Affymetrix RNA microarray is used

Affymetrix transcriptome array is used to Affymetrix used to identify upregulated genes in KD


establish a gene expression profile. patients: CD177.

Combined with with Illumina (that measure DNA The mRNA levels of CD177 were significantly higher in KD
methylation) and real-time PCR (to quantify mRNA patients than in the control groups. The CD177 values in KD
levels of CD177 and confirm Affymetrix results). patients decreased significantly after undergoing IVIG treatment.

Focused on the variations in genetic profiles


between control subjects and KD patients.

→ The chip studies consisted of 18 KD patients


that were analysed both before undergoing
intravenous immunoglobulin (IVIG) treatment and
at least 3 weeks afterward, as well as 36 non-KD
control subjects.
Trabectedin triggers direct and NK-mediated
cytotoxicity in multiple myeloma

About the article:


● Published on the 21st of March 2019 in the Journal
of Haematology and Oncology
● Authors: Pierfrancesco Tassone, Maria Cuce’, Maria
Eugenia Gallo Cantafio, and Cirrino Botta

The study shows how trabectedin, an antitumor


chemotherapy drug, can induce rapid NK mediated
lysis of cancer cells followed by the activation of
the DNA damage response.

HTA2.0 Affymetrix used for Gene expression


profiling

https://jhoonline.biomedcentral.com/track/pdf
/10.1186/s13045-019-0714-9
Multiple Myeloma (abnormal plasma cells)
Myeloma is a type of cancer affecting plasma cells in the bone marrow of multiple
parts of the body such as; the spine, pelvis rib cage etc..

It is characterized by the initial impairment of a single plasma cell and over time
the accumulation of these cancer cells can crowd out healthy white & red blood
cells in the bone marrow leading to the production of abnormal monoclonal
proteins/antibodies.
Risk factors:
Complications:
● Genetic predisposition
● Frequent infections ● Chance increases by
● Bone problems age
● Reduced kidney function https://www.mayoclinic.org/diseases-conditions/multiple-
myeloma/symptoms-causes/syc-20353378
Application of Affymetrix (DNA microarray)
in Multiple Myeloma
Method
1. Extraction of total RNA from two different samples (MM/trabectedin treated cell and healthy/non-
treated cells).
2. Synthesis of cDNA and cRNA from total RNA
3. Hybridization to genechip microarrays

*Compare findings to GEO


(Gene expression Omnibus)

List of genes included in the


DNA repair system

https://jhoonline.biomedcentral.com/track/pdf/
10.1186/s13045-019-0714-9
Findings: